Anterior gradient 2 (AGR2), a member of protein disulfide isomerase (PDI) family, is both located in cytoplasm and secreted into extracellular matrix. The orthologs of AGR2 have been linked to limb regeneration in newt and wound healing in zebrafish. In mammals, AGR2 influences multiple cell signaling pathways in tumor formation and in normal cell functions related to new tissue formation like angiogenesis. However, the function of AGR2 in mammalian wound healing remains unknown. This study aimed to investigate AGR2 expression and its function during skin wound healing and the possible application of external AGR2 in cutaneous wound to accelerate the healing process. Our results showed that AGR2 expression was induced in the migrating epidermal tongue and hyperplastic epidermis after skin excision. Topical application of recombinant AGR2 significantly accelerated wound-healing process by increasing the migration of keratinocytes (Kera.) and the recruitment of fibroblasts (Fibro.) near the wounded area. External AGR2 also promoted the migration of Kera. and Fibro. in vitro in a dose-dependent manner. The adhesion domain of AGR2 was required for the formation of focal adhesions in migrating Fibro., leading to the directional migration along AGR2 gradient. These results indicate that recombinant AGR2 accelerates skin wound healing through regulation of Kera. and Fibro. migration, thus demonstrating its potential utility as an alternative strategy of the therapeutics to accelerate the healing of acute or chronic skin wounds.
Several aminoacyl-tRNA synthetases have been reported to be overexpressed for charging essential aminoacyl-tRNAs in many cancer types. In this study, we aimed to explore the potential role of leucyl-tRNA synthetase (LARS) as an anticancer target. MTT assay was performed to screen inhibitors to human LARS (hsLARS) from compounds AN2690 and its derivatives, compounds 1–6, in U2OS and SKOV3 cells. The compound with the strongest inhibitory ability was further investigated for its inhibitory effect in cancer cell lines and in an animal tumor model. Additionally, a LARS-rescue experiment was performed to explore the potential target in U2OS using Western blot and flow cytometry. Luciferase reporter assay was designed to analyze the effect of of hsLARS inhibitor on p21 activation. We identified an hsLARS inhibitor (compound 2) that suppressed the proliferation of U2OS and SKOV3 cells in vitro. A LARS-rescue experiment demonstrated that the proliferation inhibition was induced by targeting intracellular LARS. In addition, the hsLARS inhibition was shown to activate the p21 early transcription and promote cell apoptosis, as well as reduce implanted EMT6 tumor progression in mice. Our results suggest that LARS might serve as a potential anticancer target through the p21 signaling pathway and that the nutritional signaling pathway may provide a valuable anticancer strategy for further investigation.
The effective induction of p21(WAF1/CIP1/Cdkn1a) (p21) expression in p53-negative cancer cells is an important avenue in cancer management. We investigated the ability of various common chemotherapeutic drugs to induce p21 expression in p53-negative cancer cells and showed that the induction of p21 expression by oxaliplatin is caused by the derepression of a previously unrecognized negative regulatory element with a Sp1/Sp3 palindrome sequence core at -216 to -236 of the p21 proximal promoter. Electrophoretic mobility shift and antibody super-shift assays confirmed the specific binding of Sp1/Sp3, and showed that the oxaliplatin-mediated derepression of p21 transcription was associated with an increased Sp1/Sp3 phosphorylation and binding affinity to the oxaliplatin-responsive element. A search of the ENCODE database for vertebrate-conserved genomic elements identified the Sp1/Sp3 palindrome element as the only vertebrate-conserved element within the 500-bp proximal p21 promoter region, indicating its fundamental importance. In in vivo competition assays, transfected synthetic Sp1/Sp3 palindrome elements derepressed the cotransfected or endogenous p21 promoter in a dosage-dependent manner. This derepression was not seen in oxaliplatin-treated cells, suggesting that the exogenous Sp1/Sp3 palindrome and oxaliplatin had the same downstream signaling target. Taken together, our results revealed, for the first time, this evolutionarily conserved Sp1/Sp3 palindrome element in the proximal p21 promoter that serves as a regulatory repressor to maintain p21 basal level expression.
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